Production and purification of monoclonal antibodies to Pisum and Avena phytochrome

Abstract

At least nine monoclonal antibodies against phytochrome from Pisum sativum L. and 20 against phytochrome from Avena sativa L. have been obtained from mouse hybridomas that were produced by fusion of spleen cells with SP 2/O-Ag14 myeloma cells. Hybridomas were selected and cloned in a single step by plating on a semisolid methylcellulose medium. Eight antibodies to Pisum and one to Avena phytochrome were immunopurified from hybridoma medium or ascitic fluid. When necessary, secreted antibodies were verified to be against phytochrome by demonstrating to be against phytochrome by demonstrating immunoadsorption of phytochrome, detected as loss of photoactivity and-or by appearance of the approx. 120,000-dalton phytochrome band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Phytochrome Phosphorylation Modulates Light Signaling by Influencing the Protein–Protein Interaction[W]

“…The protein bands on the SDS-PAGE gel were transferred to a polyvinylidene difluoride membrane (Hybond-P; Amersham Pharmacia Biotech), and the membrane was incubated with oat phyA-specific monoclonal antibody, oat22 or oat25 (Cordonnier et al, 1983), for 2 h and developed using an ECL protein gel blotting analysis system (Amersham Pharmacia Biotech). To assess the expression of oat phyA and S598A mutant proteins in 4-d-old dark-grown seedlings, crude extracts were purified by the EZ method as described (Martínez-García et al, 1999).…”

Phytochrome Levels in Light-Grown Avena Change in Response to End-of-Day Irradiations

“…The supplemental illumination was provided by metal halide and high-pressure sodium lamps from 6 AM to 8AM and from 6 PM to 10 PM. Not only was the day length extended to on days 8 and 9 resulted in the same outcome.…”

A Photoperiod-Insensitive Barley Line Contains a Light-Labile Phytochrome B1

“…Oat (Avena sativa L.) 22 (O-22) is a MAb directed against etiolated oat phytochrome and it specifically recognizes the 124-kD phytochrome apoprotein of phyA (Cordonnier et al, 1983). GO-5 and GO-7 are specific for a 125-and a 123-kD phytochrome molecular species, respectively, and were developed against green oat phytochrome .…”

“…The 124-kD species of phytochrome in oat, which is detected by O-22, is known to be a phyA (Cordonnier et al, 1983, whereas the 123-kD species, which is detected by GO-7, is now thought to be a phyB (Childs et al, 1997). The 125-kD species, to which GO-5 binds specifically (Wang et al, 1992), and which was thought initially not to be either a phyA, phyB, or phyC , seems likely to be a phyC (R. Wikle and M.-M. Cordonnier-Pratt, unpublished data).…”